Liver represents one of the most important targets for gene medicine applications because of the access of the transgene product to systemic circulation, and because it is the site of many metabolic genetic disorders, viral infection and malignancies. A critical barrier in realizing the potential of liver-targeted gene transfer is the development of a safe and efficient gene carrier in combination with a feasible and efficient administration route. To address this issue, we are developing a nanoparticle gene delivery system based on chitosan, which is a biodegradable, biocompatible, and structurally versatile natural polymer. Our recent 'proof-of-concept'experiments have shown that intrabiliary infusion of nanoparticles achieves high levels of transgene expression in rat liver. In this proposal, we build on this finding and hypothesize that high, persistent and liver-targeted transgene expression can be achieved by the systematic optimization of the structure and physicochemical properties of chitosan-DNA nanoparticles and the intrabiliary infusion parameters. The goal of the following set of four specific aims is to experimentally verify this hypothesis and to elucidate its underlying mechanisms: (1). To synthesize and characterize novel chitosan-DNA nanoparticles with optimal complex stability, colloidal stability and hepatocyte-targeting capability. Structure- property-transfection activity relationships will be established;(2). To optimize administration parameters for intrabiliary infusion of chitosan-DNA nanoparticles. Four leading nanoparticle candidates from Aim 1 will be used to evaluate infusion parameters that could significantly affect in vivo transgene expression through intrabiliary infusion;(3). To characterize the nanoparticle/DNA delivery process at the tissue level, at the cellular level in the liver, and at the subcellular level by quantitative PCR, immunofluorescence staining and EM. The information will provide insight to the detailed mechanism of nanoparticle transport and gene delivery through intrabiliary infusion;(4). To validate the gene delivery strategies developed in Aims 1-3 by producing two types of gene product: the systemic circulation of IFN-alpha2b in normal rat, and the correction of a congenital metabolic defect in the Gunn rat mediated by the hUGT1 A1 gene. These experiments will validate our proposed gene delivery strategy and demonstrate its broad applicability for production of proteins intended for systemic distribution and for localized liver-specific diseases.